Current antiviral drugs targeting hepatitis B virus (HBV) still face challenges in achieving functional cure for chronic hepatitis B. Therefore, it is necessary to explore new therapeutic approaches and combination therapy strategies. Anticodon engineered transfer RNA (ACE-tRNA) has been widely applied in the treatment of genetic diseases caused by nonsense mutations. However, its role in antiviral therapy has not been reported yet. In this study, ACE-tRNAs were designed targeting the highly conserved stop codon (UAG) of HBV core protein (HBc). All designed ACE-tRNAs can read through the stop codon of HBc. Among them, tRNASUAG promoted HBc phosphorylation by introducing a phosphorylatable serine into the C-terminal domain of HBc, thereby reducing HBc levels and inhibiting HBV replication by promoting the degradation of HBc through the ubiquitin-proteasome pathway. In addition, ACE-tRNA and CRISPR/Cas9 technologies can be effectively integrated through a tRNASUAG-gRNA tandem array and achieve the combined inhibition of HBV replication. This is the first study to use ACE-tRNA to promote HBc degradation by introducing an amino acid that can be post-translationally modified and subsequently inhibit HBV replication. In addition, this study presents a promising therapeutic strategy for promoting the clearance of HBV infection by integrating ACE-tRNA and CRISPR/Cas9 technologies.